Chemical pesticides have become indispensable in modern agriculture for controlling insect, plant and fungal pests. It has been estimated that, worldwide, 4 million tonnes of pesticides were applied to crops in 1999. Global pesticide sales reached approximately US$33 billion in 2004. However, the vast majority of the active ingredients do not reach target pests but instead enter groundwater and rivers through misapplication, runoff and leaching. Accordingly, there is mounting public concern about the deleterious effects of pesticide contamination, through its impacts on both the environment and on human health. Technologies for decontaminating pesticide residues are therefore needed. Traditional methods of remediation of toxic compounds including incineration, burial or chemical degradation (oxidation, reduction and hydrolysis) are often too expensive or otherwise impractical for cleaning up the pesticide residues, but these problems may be ameliorated by bioremediation, a process by which biological agents and processes are utilised to detoxify environmental pollutants. Bioremediation has been applied effectively to the clean-up of pesticides in irrigation tail water, groundwater and soil.
The direct application of live microorganisms to contaminated soils has been used to degrade a number of pesticides. An alternative to microbial remediation is the use of a relatively new technology known as enzymatic bioremediation. It is particularly suited to environments not conducive to microbial growth or situations where rapid remediation is required. This includes irrigation run-off water, spills, commodity clean-up, wash-down of farm machinery and for the personal protection of agricultural workers. Enzyme-based bioremediation often utilises the degradative capabilities of pesticide-resistant microorganisms, insects or weeds as a source of catalytic proteins. Typically, once an organism possessing the desired degradative property is isolated, gene technology is then used to clone the gene(s) responsible. The enzymatic properties of the resultant gene products are determined and, if necessary, improved using modern molecular biology techniques. These enzymes are then produced in quantity and applied directly to the affected area, e.g. field or drainage waters, in order to reduce pesticide load and, hence, toxicity. This technology is currently being applied to the clean-up of pesticide residues from agricultural wastewater.
Carbamate pesticides are derived from carbamic acid (HOOC—NH2) and possess the general structure shown in FIG. 1A. The chemical side chains principally govern the biological activity of the pesticide. The atom denoted by the X is either an oxygen or a sulfur, whereas R1 and R2 can be a number of different organic side chains, although quite often a methyl group or a hydrogen. R3 is usually a bulky aromatic group or an oxime moiety. Benzimidazole carbamate fungicides include benomyl, carbendazim, cypendazole, debacarb and mecarbinzid.
Early studies provided evidence that microorganisms play a role in enhancing carbamate degradation in “aggressive” soils in which repeated pesticide applications led to a greatly reduced persistence of these compounds (Karns et al., 1986; Derbyshire et al., 1987; Karns et al., 1991; Tomasek et al., 1989). It has since been shown that the primary step in the carbamate degradation process is often hydrolysis across the carbamate linkage (Topp et al., 1993). This simple reaction is predominantly a cofactor-independent process and thus advantageous for enzymes that are to be considered for bioremediation. Several carbamate hydrolase enzymes responsible for the degradation have now been isolated from various organisms. Examples of enzymes which have been shown to degrade carbamates include MCD (Tomasek et al., 1989), cahA (Hayatsu et al., 2001), cehA (Bomscheuer et al., 2002) and PCD (Genbank Accession No. M94965).
Involvement of fungi and bacteria in enhanced and nonenhanced biodegradation of carbendazim and other benzimidazole compounds in soil has been reported (Yarden et al., 1990). Furthermore, two carbendazim degrading bacteria, Ralstonia sp. strain 1-1 and Rhodococcus sp. Dj1-6 have been described (Zhang et al., 2005; Jing-Liang et al., 2006). However, no gene-enzyme system responsible for the carbendazim hydrolytic activity is known to date.
There is a need for methods and enzymes which can be used for the bioremediation of, for example, soils, foodstuff and water samples contaminated with toxic compounds including degrading benzimidazole carbamate fungicides, carbanilate fungicides, sulfonamide herbicides, thioamide herbicides and/or synthetic pyrethroid insecticides.